Gene expression construct

ABSTRACT

Provided are novel nucleic acid molecule encoding modified HAP1 DNA-binding domains. Also provided are novel reporter genes based on GFP and extension. The materials provided by the invention may be used in a variety of methods of activating genes having HAP1 upstream activation sequences in plants, which methods can be used to co-ordinate or investigate gene expression, optionally in conjunction with GAL4-regulated expression, and also in novel “enhancer” traps.

TECHNICAL FIELD

The present invention relates generally to methods and materials for usein achieving and detecting gene expression, particularly localisedexpression of genes in a plant, and the detection of gene expression ina plant.

PRIOR ART

Developing multicellular tissues or organs generally demonstrate acapacity for self-organisation. For example, wounded tissues generallyrespond in a robust and coordinated fashion to allow repair, and localinduction events can initiate prolonged and coordinated developmentalprocesses. These types of developmental plasticity and functionalautonomy are particularly evident in plant tissues. The basic featuresof a plant's body plan are established during embryogenesis, however itsfinal form results from the continued growth of meristems and theformation of organs throughout its life, often in a modular andindeterminate fashion. Plant cells are constrained by rigid cell wallsand are generally non-motile, so there is the clear possibility thatcell fates within a meristem are determined by lineage. However,evidence from plant chimera and wounding studies have demonstrated amore important role for cell-cell interactions during fate determination(reviewed in Steeves & Sussex, Patterns in Plant Development, 1989) andlaser ablation of cells within the Arabidopsis root meristem has shownthat after the death of a cell, a neighbouring cell can be triggered todivide and compensate for the loss (van der Berg et al., Nature378:62-65, 1995). It is likely that positional information during plantdevelopment is obtained via cell-cell contact, and that the coordinationand fate of cells within a developing meristem may be determined by anetwork of local cellular interactions. The present inventors havechosen the Arabidopsis root meristem as a model system for investigatingintercellular interactions. The root meristem possesses indeterminategrowth and has a simple and transparent architecture. Arabidopsis isgenetically amenable, and one can routinely generate transgenic linesfor work with the intact organism.

However, in order to dissect and engineer local cell-cell interactions,it is crucial that one can (i) clearly visualise individual cells insideliving meristems and (ii) have the means to perturb them. Over the pastseveral years, the present inventors have developed a set of genetic andoptical techniques which enable the manipulation and visualisation ofcells within living plants.

In order to genetically manipulate cells during meristem development,the inventors have previously devised a scheme for targeted geneexpression, which is based on a method widely used in Drosophila (Brandand Perrimon, Development 118:401-415, 1993). PCT/GB97/00406 describes amethod using a highly modified transcription factor derived from theyeast GAL4 protein to form Arabidopsis plant lines that displaylocalised expression of the foreign transcription factor, which can beused to trigger the ectopic expression of any other chosen gene at aparticular time and place during the growth of the plant. The expressionof the transcription factor can be followed using GFP as a reportergene.

However, this system has a number of problems, one of which that it islimited in its application to the activation of a single chosen gene orthe simultaneous activation of different genes within the same celltypes, but does not allow the activation of different genes in differentcell types and/or at different times within the same plant.

Thus it can be seen that another modified transcription factor whichcould be used in conjunction with or as an alternative to GAL4 wouldprovide a valuable contribution to the art.

In order to visualise plant cells in transgenic plants, the geneencoding jellyfish green fluorescent protein (GFP) has been adapted foruse as a reporter gene. The wild-type GFP cDNA is not expressed inArabidopsis. The present inventors have extensively modified the gfpgene to remove a cryptic intron, to introduce mutations that conferimproved folding and spectral properties and to alter the subcellularlocalisation of the protein. All of these alterations have beenincorporated into a single modified form of the gene (mgfp5-ER) whichcan be routinely used for monitoring gene expression and marking cellsin live transgenic plants (Siemering et al., Current Biology6:1653-1663, 1996; Haseloff et al., PNAS 94:2122-2127, 1997).

Fluorescence microscopy techniques for high resolution observation ofliving cells have been developed. The expression of GFP within anorganism produces an intrinsic fluorescence that colours normal cellularprocesses, and high resolution optical techniques can be usednon-invasively to monitor the dynamic activities of these living cells.Using coverslip-based culture vessels, specialised microscope objectivesand the optical sectioning properties of the confocal microscope, it ispossible to monitor simply and precisely both the arrangement of livingcells within a meristem, and their behaviour through long time-lapseobservations. Further, the present inventors have recently constructedcyan and yellow emitting GFP variants that can be distinguished from thegreen fluorescent protein during confocal microscopy. These colourvariants have enabled simultaneous imaging of different tagged proteinsin living cells (Haseloff, J., “GFP variants for multispectral imagingof living cells”, in Methods in Cell Biology, Vol. 58, Kay, S. andSullivan, K. Eds. Academic Press (1999).

However, the usefulness of reporter proteins such as GFP is limited bythe fact that, when plant tissues are cleared and stained for detailed3-Dimensional analysis, reporter proteins such as GFP are lost from thetissues.

Thus it can be seen that a robust insoluble reporter protein wouldprovide a valuable contribution to the art.

DISCLOSURE OF THE INVENTION

In a first aspect of the present invention there is provided an isolatednucleic acid, expressible in a plant cell, encoding at least aneffective portion of a HAP1 DNA-binding domain, wherein the sequence hasan A/T base content substantially reduced compared to the wild-typesequence.

“Effective Portion”

An effective portion of the DNA-binding domain is a portion sufficientto retain most (i.e. over 50%) of the DNA-binding activity of the fulllength DNA-binding domain. Preferably the “effective portion” comprisesamino acid residues 1 to 94 of the yeast polypeptide, which we havefound to be the minimal amount required to retain DNA binding activity.Typically, the “effective portion” will comprise at least 60% of thefull-length sequence of the DNA-binding domain.

“A/T Base Content”

The A/T content of the wild-type yeast sequence encoding the DNA-bindingdomain of HAP1 is about 54%. The % A/T base content of the sequence ofthe invention encoding the effective portion of the HAP1 should be takento be substantially reduced when it is less than 45%. Preferably, itwill be less than 40%, most preferably it is 39%.

Preferably, the encoded polypeptide will have the identical amino acidsequence to the wild-type HAP1 polypeptide shown in FIG. 2 b (bottomline). However, on the other hand, the encoded polypeptide may comprisean amino acid sequence which differs by one or more amino acid residuesfrom the amino acid sequence shown in FIG. 2 b (bottom line).

Nucleic acid encoding at least an effective portion of a HAP1DNA-binding domain which is an amino acid sequence mutant, variant orderivative of the amino acid sequence shown in FIG. 2 b, wherein thenucleic acid sequence has an A/T base content substantially reducedcompared to the wild-type sequence is therefore included within thescope of the present invention.

A peptide which is an amino acid sequence variant, derivative or mutantof an amino acid sequence of a peptide may comprise an amino acidsequence which shares greater than about 60% sequence identity with thesequence of the amino acid sequence shown in FIG. 2 b (bottom line),greater than about 70%, greater than about 80%, greater than about 90%or greater than about 95%. The sequence may share greater than about 70%similarity, greater than about 80% similarity, greater than about 90%similarity or greater than about 95% similarity with the amino acidsequence shown in FIG. 2 b (bottom line).

For amino acid “homology”, this may be understood to be similarity(according to the established principles of amino acid similarity, e.g.as determined using the algorithm GAP (as described below) or identity.

Amino acid similarity is generally defined with reference to thealgorithm GAP (Genetics Computer Group, Madison, Wis.). GAP uses theNeedleman and Wunsch algorithm to align two complete sequences thatmaximizes the number of matches and minimizes the number of gaps.Generally, the default parameters are used, with a gap creationpenalty=12 and gap extension penalty=4. Use of GAP may be preferred butother algorithms may be used, e.g. BLAST (which uses the method ofAltschul et al. (1990) J. Mol. Biol. 215: 405-410), FASTA (which usesthe method of Pearson and Lipman (1988) PNAS USA 85: 2444-2448), or theSmith-Waterman algorithm (Smith and Waterman (1981) J. Mol. Biol. 147:195-197), generally employing default parameters.

Similarity allows for “conservative variation”, i.e. substitution of onehydrophobic residue such as isoleucine, valine, leucine or methioninefor another, or the substitution of one polar residue for another, suchas arginine for lysine, glutamic for aspartic acid, or glutamine forasparagine.

In a preferred embodiment, the nucleic acid of the invention comprisesthe nucleic acid sequence of modified HAP1 (labelled mHAP1—middle lineof FIG. 2 b), which sequence has a substantially reduced A/T contentrelative to the wild-type yeast sequence which is shown in the top lineof FIG. 2 b, labelled HAP1 (substantially reduced base content isdefined above).

In a preferred embodiment, the nucleic acid encoding the portion of HAP1binding domain is fused to a nucleic acid sequence, said sequence beingstructural (e.g. encoding functional polypeptides) and/or regulatory.Preferably, said sequence encodes a transcriptional activator. Thetranscriptional activator may be the activation domain of the HAP1protein, in which case the sequence encoding the HAP1 transcriptionalactivator should be optimised for expression in plants, by, for example,reducing the A/T content thereof. Alternatively, the transcriptionalactivator may be any transcriptional activator known by the skilledperson to be active in plants. In such cases, the sequence of theinvention thus encodes a chimeric polypeptide. In a preferredembodiment, the transcriptional activator domain is that of the herpessimplex virus (HSV) VP-16 (Greaves and O'Hare, J. Virol 63 1641-1650,(1989)). Preferably, the sequence comprises the nucleic acid sequence ofVP16 shown in FIG. 2 c (from nucleotide 293 onwards, i.e., the part ofthe top line of sequence which is in upper case). Thus in a preferredembodiment of the invention, there is provided a chimeric polypeptidecomprising the nucleic acid sequence of the mHAP1-VPI6 chimera shown inFIG. 2 c (i.e., the entire nucleotide sequence of FIG. 2 c (top line)).

Other suitable transcriptional activation domains include certainpeptides encoded by the E. coli genomic DNA fragments (Ma and Ptashne,Cell 51 113-119 (1987)) or synthetic peptides designed to formamphiphilic α-helix. (Giniger and Ptashne Nature 330 670-672 (1987)). Acommon requirement for suitable transcriptional activation domains isthe need for excess charge (Gill and Ptashne, Cell 51 113-119 (1987),Estruch et al Nucl. Acids Res. 22 3983-3989 (1994)). Using thiscriteria, the skilled person is able to select or synthesise sequenceswhich encode transcriptional activation activity in plants.

In a further aspect of the present invention, there is provided anucleic acid construct, comprising the nucleic acid defined above.

Preferred Vectors

In one aspect of the present invention, the nucleic acid construct is inthe form of a recombinant and preferably replicable vector. “Vector” isdefined to include, inter alia, any plasmid, cosmid, phage orAgrobacterium binary vector in double or single stranded linear orcircular form which may or may not be self transmissible or mobilizable,and which can transform a prokaryotic or eukaryotic host either byintegration into the cellular genome or exist extrachromosomally (e.g.autonomous replicating plasmid with an origin of replication).

Generally speaking, those skilled in the art are well able to constructvectors and design protocols for recombinant gene expression. Suitablevectors can be chosen or constructed, containing appropriate regulatorysequences, including promoter sequences, terminator fragments,polyadenylation sequences, enhancer sequences, marker genes and othersequences as appropriate. For further details see, for example,Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al,1989, Cold Spring Harbor Laboratory Press or Current Protocols inMolecular Biology, Second Edition, Ausubel et al. eds., John Wiley &Sons, 1992.

A vector including nucleic acid according to the present invention neednot include a promoter or other regulatory sequence, particularly if thevector is to be used to introduce the nucleic acid into cells forrecombination into the genome.

Preferably the nucleic acid in the vector is under the control of, andoperably linked to, an appropriate promoter or other regulatory elementsfor transcription in a host cell such as a microbial, e.g. bacterial, orplant cell. The vector may be a bi-functional expression vector whichfunctions in multiple hosts. In the case of genomic DNA, this maycontain its own promoter or other regulatory elements and in the case ofcDNA this may be under the control of an appropriate promoter or otherregulatory elements for expression in the host cell.

By “promoter” is meant a sequence of nucleotides from whichtranscription may be initiated of DNA operably linked downstream (i.e.in the 3′ direction on the sense strand of double-stranded DNA).

“Operably linked” means joined as part of the same nucleic acidmolecule, suitably positioned and oriented for transcription to beinitiated from the promoter. DNA operably linked to a promoter is “undertranscriptional initiation regulation” of the promoter.

Thus this aspect of the invention provides a gene construct, preferablya replicable vector, comprising a promoter operably linked to a nucleicacid provided by the present invention, for example, the sequenceencoding the HAP1 DNA-binding domain.

Particularly of interest in the present context are nucleic acidconstructs which operate as plant vectors. Specific procedures andvectors previously used with wide success upon plants are described byGuerineau and Mullineaux (1993) (Plant transformation and expressionvectors. In: Plant Molecular Biology Labfax (Croy R R D ed) Oxford, BIOSScientific Publishers, pp 121-148). Suitable vectors may include plantviral-derived vectors (see e.g. EP-A-194809).

Preferred Promoters

Preferably the promoter to be used in the construct is an “enhancerdependent” (or naïve) promoter, which requires the presence of asuitable enhancer sequence and appropriate transcription factors tocause substantial levels of transcription. Such naïve promoterscorrespond to the TATA box region of known plant promoters. “Plantpromoters” should be understood to refer to promoters (e.g. viral orbacterial) active in a plant cell. The naïve promoter is in competentrelationship with the sequence encoding the HAP1 DNA binding domain andtranscription activation domain such that if the promoter is insertedinto a plant host cell genome in functional relationship with anenhancer sequence and required transcription factors, the promoter willdirect expression of the HAP1 DNA-binding domain in a tissue specificmanner.

Reporter Genes

In preferred embodiments of the invention, a reporter gene operablylinked to a HAP1 upstream activation sequence (UAS) is provided, suchthat the reporter gene will be expressed in response to synthesis of thetranscriptional activator discussed above. The reporter gene may bepresent as part of a nucleic acid construct comprising the nucleic acidencoding an effective portion of a HAP1 DNA-binding domain.Alternatively, the reporter gene may be present in another nucleic acidconstruct.

The reporter gene may be any suitable reporter gene known to the skilledperson as being active in plants. Use of a reporter gene facilitatesdetermination of the UAS activity by reference to protein production.The reporter gene preferably encodes an enzyme which catalyses areaction which produces a detectable signal, preferably a visuallydetectable signal, such as a coloured product. Many examples are known,including β-galactosidase and luciferase. β-galactosidase activity maybe assayed by production of blue colour on substrate, the assay being byeye or by use of a spectro-photometer to measure absorbance.Fluorescence, for example that produced as a result of luciferaseactivity, may be quantitated using a spectrophotometer. Radioactiveassays may be used, for instance using chloramphenicolacetyltransferase, which may also be used in non-radioactive assays. Thepresence and/or amount of gene product resulting from expression fromthe reporter gene may be determined using a molecule able to bind theproduct, such as an antibody or fragment thereof. The binding moleculemay be labelled directly or indirectly using any standard technique.

Those skilled in the art are well aware of a multitude of possiblereporter genes and assay techniques which may be used to determine UASactivity. Any suitable reporter/assay may be used and it should beappreciated that no particular choice is essential to or a limitation ofthe present invention.

In preferred embodiments of the invention, the reporter gene is GFP. Thegfp gene may be the wild-type Aequorea victoria gene, or may be modifiedin any conventional way. For example, in a preferred embodiment, the gfpgene is mgfp5-ER, which has a cryptic intron removed, and has mutationswhich confer improved folding and spectral properties and alteredsubcellular localisation of the protein (Siemering et al., CurrentBiology 6:1653-1663, 1996; Haseloff et al., PNAS 94:2122-2127,1997).

However, although GFP (wild-type and modified forms) provides aconvenient method of marking cells, clearing and staining of planttissues normally result in loss of the green fluorescent protein fromthe tissues. For example, after treatment with even gentle clearingagents such as 50% ethanol or 50% glycerol, the protein becomesdislodged from treated cells.

The present inventors have overcome this problem by developing a newrobust surface marker for visualisation of plant cells utilising GFPfused to an extensin protein (see below). Thus, in preferred embodimentsof the invention, the reporter gene comprises the GFP extensin reportergene fusion as described below.

Host Plants

In a further aspect of the invention, the invention provides a plant,plantlet or part thereof (e.g. a plant host cell or cell line)comprising a nucleic acid construct of the invention.

In preferred embodiments, the construct will have become stablyintegrated into a plant cell genome. In particular, the inventionprovides a plurality of plants, plantlets or parts thereof comprising alibrary, each plant or part thereof comprising a stably maintainednucleic acid sequence encoding an effective portion of the HAP1DNA-binding domain as defined above. Preferably, the nucleic acidconstruct will be incorporated into the genome of plant cells present inthe library. The library may be of any plant of interest to the skilledperson. Suitable plants include maize, rice, tobacco, petunia, carrot,potato and Arabidopsis. Where the term “plant” is referred tohereinafter, unless context demands otherwise, it will be understoodthat the invention applies also to plantlets or parts of plants.

Each plant, plantlet or part thereof may have a particular pattern ofexpression of the integrated reporter gene. Thus, introduction of afurther gene, having a HAP1-responsive UAS into the cells will result inthe expression of the introduced gene in the same temporal/spatialpattern as the reporter gene, enabling expression of a gene of interestin selected tissues and/or at selected times.

Uses of Constructs of the Invention

In a preferred embodiment of the invention, the nucleic acid constructcan be used in an “enhancer trap assay” to identify plant enhancersequences (Sundaresan et al, Genes and Dev. 9 1797-1810). In such cases,the nucleic acid construct will preferably comprise right and leftTi-DNA, to enable random, stable insertion into the genome of a planthost cell.

As well as nucleic acid constructs for use in “enhancer trap assays”,the invention further provides a method of identifying a plant“enhancer” nucleic acid sequence, comprising the steps of:

-   -   transforming a plant cell host with a nucleic acid construct        comprising a naïve promoter sequence and a sequence encoding an        effective portion of an HAP1 binding domain fused to a        transcription activating domain, under the control of said naïve        promoter sequence,    -   wherein, when said plant is transformed with said construct such        that the promoter is in functional relationship with a host cell        enhancer sequence, the promoter will direct expression of said        HAP1 binding domain operably linked to a transcription        activating domain in the presence of “enhancer” transcription        factors.

Thus the expression of said HAP1 binding domain operably linked to atranscription activating domain will indicate the presence of such an“enhancer” sequence. Optionally, the method includes the further step ofidentifying the position and nucleic acid sequence of the enhancersequence.

For example, this may be performed by standard inverse PCR (I-PCR) orTAIL-PCR amplification of flanking sequences (see Sambrook & Russell,Molecular Cloning: a laboratory manual. 3^(rd) edition, CSHL press 2001:sections 4.75 (TAIL-PCR), 8-81 (I-PCR)).

The nucleic acid construct of the invention may thus also be used tocontrol expression of an heterologous gene in a plant or part thereof.Thus in a further aspect of the present invention, there is provided amethod of controlling expression of a gene of interest in a plant orpart thereof comprising the steps of:

-   -   introducing the gene of interest into a plant or part thereof,        said gene of interest having an HAP1 responsive upstream        activation sequence,    -   said plant or part thereof comprising a nucleic acid sequence        encoding an effective portion of an HAP1 binding domain fused to        a transcription activating domain, under the control of said        naïve promoter such that expression of a transcriptional        activator from said sequence is limited to those cell types in        which a naïve promoter sequence is in functional relationship        with a host cell enhancer sequence;    -   wherein binding of said transcriptional activator to said        upstream activator sequence causes transcriptional activation of        the gene of interest.

The nucleic acid of the invention enables the activation of differentgenes of interest in different cell types and/or at different timeswithin the same plant, particularly wherein the nucleic acid encoding atleast an effective portion of a HAP1 DNA-binding domain is used inconjunction with nucleic acid encoding a modified GAL4 transcriptionfactor, for example that described in WO97/30164, the expression of eachgene of interest being under the operable control of a differenttranscription factor.

Thus, included within the scope of the present invention is a method ofindependently controlling expression of a first and a second gene ofinterest in a plant comprising the steps of:

-   -   introducing the first gene of interest into a plant or part        thereof, said first gene of interest having an HAP1 responsive        upstream activation sequence;    -   introducing the second gene of interest into a plant or part        thereof, said second gene of interest having a GAL4 responsive        upstream activation sequence;    -   said plant or part thereof comprising a first nucleic acid        sequence, which encodes a HAP1 transcriptional activator and a        second nucleic acid sequence, which encodes a GAL4        transcriptional activator;    -   wherein binding of said HAP1 transcriptional activator to said        upstream activator sequence causes transcriptional activation of        the first gene of interest and binding of said GAL4        transcriptional activator to said upstream activator sequence        causes transcriptional activation of the second gene of        interest.

In this way, where the expression of the HAP1 transcriptional activatorand the GAL4 transcriptional activator are each independently under thecontrol of a different naïve promoter sequence, expression of eachtranscriptional activator is limited to those cell types in which thenaïve promoter sequence is in functional relationship with a host cellenhancer sequence.

Moreover, using the nucleic acid construct of the invention, thesimultaneous expression of a number of genes of interest may becontrolled. Thus, in a further aspect of the invention, there isprovided a method of co-ordinating the expression of a plurality ofgenes of interest in a plant or part thereof, comprising the steps ofintroducing the genes of interest into a plant or part thereof,

-   -   said genes of interest each being under the control of an HAP1        responsive upstream activation sequence and said plant or part        thereof comprising a nucleic acid sequence of the invention        capable of expressing an HAP1 transcriptional activator,    -   wherein binding of said HAP1 transcriptional activator to said        upstream activator sequence causes transcriptional activation of        the genes of interest.

The plurality of genes may all be associated with a single UAS, whichfacilitates their introduction into the plant or part thereof.Alternatively, one or more genes may be operably linked to a respectiveUAS.

Using the nucleic acid construct of the invention in conjunction with anucleic acid construct encoding a GAL4 transcriptional activator, theexpression of a first group of genes may be coordinated and theexpression of a second group of genes may be co-ordinated, wherein eachgroup of genes is expressed in different cell-types and/or at differenttimes.

The gene or genes of interest may be any target gene or genes, theexpression of which the researcher wishes to study. In preferredembodiments, the gene or genes of interest may be developmental genes ormay encode one or more toxins. For example, a gene of interest mayencode a toxin such as the A-chain of diphtheria toxin (DTA) and thusthe method may be used to kill specific cells, for example, within theroot meristem. Other genes of interest may encode one or more cell cycleregulatory proteins, in which case expression of such gene or genes maybe used to drive misexpression of such proteins and activate or inhibitparticular cell divisions e.g. within the root meristem. The gene orgenes of interest may encode homeodomain proteins and thus the effect oftheir ectopic expression on cell fate determination may be studied.

The gene of interest may be of unknown function. Using the methods ofthe invention, the function of a gene of interest may be determined bycomparing the phenotype of plants, or parts thereof in which the gene ofinterest is expressed with the phenotype of plants or parts thereof inwhich it is not expressed. Thus the invention extends to a method ofdetermining the function of a gene of interest comprising the steps of:

-   -   introducing a gene of interest into a plant or part thereof,        said gene of interest having an HAP1 responsive upstream        activation sequence;    -   said plant or part thereof comprising a nucleic acid sequence,        which encodes a HAP1 transcriptional activator;    -   wherein binding of said HAP1 transcriptional activator to said        upstream activator sequence causes transcriptional activation of        the gene of interest;    -   comparing the phenotype of said plant or part thereof in which        said gene of interest is expressed with a second plant or part        thereof in which said gene of interest is not expressed.

The gene or genes of interest may be “introduced” into the plant or partthereof using any conventional technique, for example, using any one ofthe vectors described above. Conveniently, the gene of interest isintroduced using Agrobacterium mediated transformation.

In preferred embodiments of the methods of the invention, a reportergene having an HAP1 responsive upstream activation sequence is provided,such that binding of said transcriptional activator to said upstreamactivator sequence causes transcriptional activation of the reportergene. The reporter gene may be any suitable reporter gene, details ofwhich are given above. Preferably, the reporter gene will be theextensin-GFP fusion gene described below.

Extensin-GFP Reporter Gene Construct

As described above, conventional cell markers utilising GFP suffer fromthe disadvantage that, during clearing of the tissues, the GFP is oftenlost to at least some degree. The present inventors have overcome thisproblem by developing a new robust surface marker for visualisation ofplant cells utilising GFP.

This aspect of the invention is based on the inventors' demonstrationthat, when a coding sequence encoding GFP is fused to the codingsequence of the carrot extensin gene, the resulting expressedextensin-GFP fusion protein results in a bright marker resistant toclearing techniques which normally result in complete loss of GFP fromtreated tissues. Thus in a preferred embodiment of the presentinvention, the reporter gene construct is an extensin-GFP reporter gene.

Indeed the extensin-GFP reporter gene fusion forms a separate aspect ofthe present invention. Thus, this aspect of the present inventionprovides a gene fusion, expressible in a plant cell, comprising anucleic acid sequence encoding a green fluorescent protein operablylinked to a nucleic acid sequence encoding at least an effective portionof extensin.

An effective portion of extensin is a portion sufficient to retain most(i.e. over 50%) of the activity of the full length carrot extensin.Typically, the “effective portion” will comprise at least 60% of thefull-length sequence of the carrot extensin. Wild-type extensin isinvolved in cell wall expansion in plants, other cell wall expansionproteins may be used, as would be understood by the person skilled inthe art.

In a preferred embodiment, the nucleic acid sequence encoding the greenfluorescent protein is the nucleic acid sequence of GFP shown in FIG. 3b (uppercase nucleic acid sequence) and/or the nucleic acid sequenceencoding the extensin protein is the nucleic acid sequence of extensinshown in FIG. 3 b (lowercase amino acid sequence). The gene fusionpreferably comprises the a nucleic acid molecule having the entiresequence shown in FIG. 3 b.

In one aspect of the present invention, the gene fusion is in the formof a recombinant and preferably replicable vector. Details of suitablevectors are those described above for the nucleic acid construct of theinvention.

The extensin-GFP gene fusion is preferably part of a construct in whichit is in operable relationship with an upstream activation sequence orpromoter, the activation of which by an activation domain of atranscription factor causes expression of the extensin-GFP gene fusion.

In a further aspect of the invention, the invention provides a plant,plantlet or part thereof (e.g. a plant host cell or cell line)comprising the extensin-GFP gene fusion of the invention. In preferredembodiments, the extensin-GFP gene fusion will have become stablyintegrated into a plant cell genome.

Uses of Extensin-GFP Gene Fusion and Fusion Protein

The extensin-GFP gene fusion may be used in any of the applications forwhich a reporter gene may be routinely used, including those describedabove for “Reporter Genes”.

In particular, the extensin-GFP gene fusion may be used to visualisespecific patterns of cell-wall-localised expression in assays andmethods as described herein.

Thus, for example, the invention provides the use of the extensin-GFPgene fusion of the invention in an enhancer trap assay. In a preferredembodiment, the “enhancer trap” assay is the “enhancer trap” assaydescribed above. In such an assay, the extensin-GFP gene fusion is inoperable relationship with an upstream activation sequence or promoter,the activation of which by an activation domain of an HAP1 bindingdomain operably linked to a transcription activating domain causesexpression of the extensin-GFP gene fusion and thus enables thevisualisation of expression of the HAP1 transcription factor.

Further included within the scope of the invention is the use of theextensin-GFP gene fusion in a method of cell sorting including the stepof screening plants, plantlets, parts or cells thereof for extensin-GFPexpression and selecting those plants, plantlets, parts or cells thereofwhich express GFP-extensin-GFP.

The cells expressing the extensin-GFP protein may be isolated usingmethods known to the skilled person, e.g. using antibody based sortingmethods. For example, to isolate cell-types expressing extensin-GFPprotein in a plant, e.g. Arabidopsis, from those not expressing theextensin-GFP protein, tissues from the transgenic plant may be treatedwith one or more enzymes e.g. pectinase, to liberate cells, followed byincubation with anti-GFP antibody coated magnetic particles. The purityof the magnetically isolated cells may be checked by fluorescencemicroscopy.

The same technique could be useful for studying the protein componentsof specific cell types, for example using antibody assays or fluorescent2D gel display techniques. If unfixed cells are used, biochemicalactivities may be assayed. In addition, it is envisaged that sequentialselection for different epitopes may be used to isolate cellularsubpopulations. For example, if a cell wall GFP marker provided anepitope for the selection of certain cell types, a second independentmarker could be used to select an even more specific sub-population(e.g. using a marker for a natural cell wall component).

Screening for Reporter Gene Expression in Plants

Expression of a reporter gene may be monitored using any suitabletechnique known to the person skilled in the art. For example, for thescreening of shoots and roots of transgenic plantlets in which thereporter protein is a fluorescent protein such as GFP or theextensin-GFP of the invention, expression can be screened directly usingepifluorescence microscopy, to, for example, monitor expression indeveloping meristems.

For the monitoring of expression of fluorescent proteins, multispectraldynamic imaging may be used. Such confocal microscope based methodsallow high resolution observation of living cells. The expression of GFPwithin an organism produces an intrinsic fluorescence that coloursnormal cellular processes, and high resolution optical techniques can beused non-invasively to monitor the dynamic activities of these livingcells. Using coverslip-based culture vessels, specialised microscopeobjectives and the optical sectioning properties of the confocalmicroscope, it is possible to monitor simply and precisely both thearrangement of living cells within a meristem, and their behaviourthrough long timelapse observations (seehttp://www.plantsci.cam.ac.uk/Haseloff). Further, the use of cyan andyellow emitting GFP variants that can be distinguished from the greenfluorescent protein during confocal microscopy enable simultaneousimaging of different tagged proteins in living cells.

As a further or alternative screen, a second screen may be used on adulttransgenic plants, in which parts of the plants such as the flowers orsiliques are dissected and the fluorescence of parts monitored. Suchscreens are particularly useful for identifying expression patterns inembryos and floral parts, in which GFP may not be expressed inplantlets.

The invention will now be further described with reference to thefollowing non-limiting Figures and Examples. Other embodiments of theinvention will occur to those skilled in the art in the light of these.

FIGURES

FIG. 1 shows oligonucleotides for construction of mHAP1 DNA bindingdomain.

FIG. 2 a shows a schematic diagram of the mHAP1-VP16 synthetictranscription activator chimeric gene

FIG. 2 b shows the nucleic acid sequence of wild-type HAP1 (topline—labelled HAP1); the nucleic acid sequence of modified HAP1 (middleline—labelled mHAP1); and the encoded amino acid sequence (bottom line)

FIG. 2 c shows the nucleic acid sequence (top line) of the mHAP1-VP16synthetic transcription activator chimeric gene, in which the HAP1sequence is the modified sequence (running from the 5′ terminus toposition 292); the VP19 nucleic acid sequence runs from position 293onwards and is shown in upper case); and the amino acid sequence of thesynthetic transcription activator chimeric protein is shown in thebottom line.

FIG. 3 a shows a schematic diagram of the extensin-GFP gene fusion.

FIG. 3 b shows the coding sequence of the extensin-GFP fusion of FIG. 3a in the top line and the encoded amino acid sequence in the bottomline. Extensin nucleotide sequence is shown in lower case and the GFPnucleotide sequence is shown in uppercase.

FIG. 4 shows expression of an extensin-GFP gene fusion in transgenicArabidopsis. A 35S-extensin-GFP construction was introduced intoArabidopsis using Agrobacterium mediated transformation. Confocaloptical sections of transformed plantlets are shown. Chlorophyllautofluorescence is seen in the red channel.

FIG. 5 shows oligonucleotides used in construction of a HAP1 DNA bindingsite.

EXAMPLES Example 1 Construction of the Modified HAP1-VP16 Gene

The yeast HAP1 protein is a member of a family of zinc-finger (Cys₄)transcription factors which are limited to fungi, and homologues havenot been found in plants to date. Yeast genes have a high A/T contentand are often poorly expressed in Arabidopsis due to aberrantpost-transcriptional processing. A synthetic gene which has an elevatedG/C content, and in which the DNA binding domain is fused to the highlyactive and G/C-rich transcription activator domain of VP16, wasconstructed:

Three long oligonucleotides, mHAP1-A, B & C (shown in FIG. 1) were madeusing automated synthesis on solid supports. The oligonucleotidesencoded the predicted DNA binding domain of HAP1 protein with modifiedcodon usage. Codon usage was modified according to the followingcriteria:

-   (i) GC content was increased-   (ii) splice junction consensus sequences were avoided-   (iii) the resultant amino acid sequence encoded by the nucleic acid    was unchanged.

Oligonucleotides mHAP1-D and E (FIG. 1) contained complementary sequencecorresponding to the junctions of the three longer oligonucleotides. Thesynthetic oligonucleotides were purified by polyacrylamide gelelectrophoresis and phosphorylated after incubation with ATP and T4polynucleotide kinase. The oligonucleotides were then mixed and heatedat 94° C. for 1 min and annealed at 60° C. for 5 min. After cooling, thesample was treated with T4 DNA ligase to produce a small quantity ofsingle-strand DNA corresponding to the mHAP1 DNA binding domain. Thiswas then used as a template for PCR amplification with oligonucleotidesmHAP1-5′ and 3′ (FIG. 1).

FIG. 2 a shows in diagrammatic form the mHAP1-VP16 synthetictranscription activator chimeric gene (FIG. 2 c entire sequence). TheDNA sequence, encoding, in the 5′ portion (bases 1 to 292), the modifiedHAP1 DNA binding domain (see also FIG. 2 b middle sequence) andencoding, in the 3′ portion (bases 293-533), the transcriptionalactivation domain from HSV VP16, is shown as the top sequence of FIG. 2c with the encoded amino acid sequence shown below. The SacI restrictionendonuclease site within the gene is marked.

The wild-type sequence of the HAP1 binding domain (see the top sequenceof FIG. 2 b) is shown above for comparison of the A/T %. The wild-typeHAP1 DNA binding domain DNA sequence is A/T rich.

Example 2 Construction of Insoluble GFP Marker

A variant of GFP was fused to the coding sequence of a carrot extensin.PCR amplification was used to obtain a copy of the extensin gene,isolated from carrots purchased in Cambridge market square. The carrotgene was genetically fused to a variant of green fluorescent proteinobtained from Packard Biosciences (Meridian, Conn. (GFPemd).

A schematic diagram of the extensin-GFP gene fusion is shown in FIG. 3 awith the coding sequence shown in FIG. 3 b (top line), the encoded aminoacid sequence is shown below in the bottom line of FIG. 3 b. Extensinsequence is in lower case and GFP sequence is in uppercase.

Example 3 Expression of Extensin GFP Gene Fusion in TransgenicArabidopsis

a) Construction of the 35S-Extensin-GFP Construct.

A variant of GFP was fused to the coding sequence of a carrot extensin.PCR amplification was used to obtain a copy of the gene, isolated fromcarrots purchased in Cambridge market square. The carrot gene wasgenetically fused to a variant of green fluorescent protein obtainedfrom Packard Bioscience (GFPemd (emerald)). Expression of this genefusion in transgenic Arabidopsis tissues results in the decoration ofcell walls with bright fluorescence.

Construction of the GFP-Extensin Gene

The following oligonucleotides were synthesised, and used as primers forthe PCR amplification of the carrot extensin gene. CarExt5 GGC GGA TCCAAC AAT GGG AAG AAT TGC TAG AGG CTC CarExt3 GGC GGA TTC GTA GTG GTG AGGAGG AGG AGG TGA CGT

Template carrot DNA was isolated using a Qiagen DNA extraction kit(UK—QIAGEN Ltd., Boundary Court, Gatwick Road, Crawley, West Sussex,RH10 9AX), and 1 microgram of isolated carrot DNA was used in a PCRreaction with VENT polymerase (New England Biolabs), (30 cycles: 92° C.30 sec, 60° C. 30 sec, 72° C. 60 sec). The amplified product waspurified by 1% agarose gel electrophoresis, and digested with therestriction endonucleases BamH1 and EcoR1. The cut fragment was thenligated into a plasmid vector that contained a GFP gene with an EcoR1restriction fused to the N-terminus of the coding sequence, Haseloff,J., Siemering, K. R., Prasher, D. C. and Hodge, S Removal of a crypticintron and subcellular localization of green fluorescent protein arerequired to mark transgenic Arabidopsis plants brightly. Proc. Natl.Acad. Sci. USA. 94, 2122-2127 (1997). The resulting plasmid contained atranslational fusion between carrot extensin and the GFPemd gene(Packard Bioscience).

FIG. 3 a shows a schematic diagram of the extensin-GFP gene fusion. Theextensin sequences lie between the BamH1 and EcoR1 sites, and the GFPsequences lie between the EcoR1 and Sac1 sequences.

This reporter gene has been tested by insertion into planttransformation vectors, and expressed in transgenic Arabidopsis plantsbehind a constitutive CaMV 35S promoter, and as part of a HAP1-basedenhancer trap vector. Bright, cell wall localised fluorescence resultsin both cases.

b) Transformation of Arabidopsis thaliana

Arabidopsis thaliana was transformed using the method given inPCT/GB97/00406.

Expression of the extensin-GFP gene fusion in transgenic Arabidopsistissues results in the decoration of cell walls with bright fluorescence(FIG. 4). Extensin becomes covalently linked to the cell wall matrix,and the GFP-extensin marker is resistant to various clearing techniquesthat normally result in complete loss of the protein from treatedtissues. For example, the cell wall bound signal is retained afterglycerol infiltration.

Example 4 Construction of a HAP1 Promoter for Use in Plants

An optimised multimeric binding site for HAP 1 was synthesised andcloned behind a GFP promoter. Oligonucleotides used in the constructionof the binding site are shown in FIG. 5 (UASHAP1a and UASHAP1b).

The oligonucleotides were phosphorylated using polynucleotide kinase,annealed, and ligated into the HinD III-Xba I sites of a UAS_(GAL4)containing vector. The oligonucleotide sequences replaced the UAS_(GAL4)with the appropriate UAS_(HAP1) sequences—already positioned upstream ofa plant TATA box and GFP reporter gene.

Example 5 Construction of HAP1-GFP Enhancer Trap Vector

An enhancer trap vector was constructed using the modified HAP1-VP16gene positioned with a minimal (naïve) promoter and the extensin GFPgene fusion as described above.

The PCR product produced as described in Example 1 was cut with BamH1and Sac1 restriction endonucleases and purified after electrophoresisthrough a 1.5% LGT agarose gel.

The plasmid pCMVGal65 (Cousens et al., EMBO J. 8:2337-2342, 1989) wasused as a source of the VP16 sequence. A SacI-KpnI fragment, whichencodes the activation domain of the herpes simplex virus VP16 protein,had been previously fused to a modified form of the GAL4 DNA bindingdomain (Haseloff and Hodge, U.S. Pat. No. 6,255,558 B1) within a plantenhancer-trap vector, pET-15 (GAL4-GFP). The GAL4 sequence was excisedfrom the pET-15 vector by restriction endonuclease digestion with BamH1and Sac1, and replaced by ligation with the amplified mHAP1 sequence(see construction of extensin-GFP gene).

The mHAP1-VP16 gene was directly assayed for activity in transformedArabidopsis plants by Agrobacterium-mediated transformation (Valvekenset al. Proc. Natl. Acad. Sci. USA 85:5536-5540, 1988).

Example 6 Enhancer Trap Screen

The vector was used to transform Arabidopsis thaliana usingAgrobacterium mediated transformation as described in Example 3. In thisway, large numbers of transgenic calli are regenerated, induced to formroots and shoots and are directly screened by epifluorescence microscopyfor extensin-GFP expression in the developing meristems.

A suitable protocol is as follows:

(1) 20-100 transgenic Arabidopsis seed were placed in a 1.5 ml microfugetube and washed for about 1 min with 1 ml of ethanol.

(2) Seeds were then incubated with 1 ml of a surface sterilisingsolution containing 1% (w/v) sodium hypochlorite and 0.1% (v/v) NP40detergent, for 15 min at room temperature.

(3) The seeds were then washed three times with 1 ml of sterile water,and transferred by pipette to agar plates containing GM medium(Valvekens, D., Van Montagu, M and Van Lijsebettens, M. (1988)Agrobacterium tumefaciens-mediated transformation of Arabidopsisthaliana root explants by using kanamycin selection. Proceedings of theNational Academy of Sciences U.S.A. 85:5536-5540). 1x Murashige andSkoog basal medium with Gamborgs B5 vitamins (Sigma) 1% sucrose 0.5 g/l2-(N-morpholino)ethanesulfonic acid (MES) 0.8% agar (adjusted to pH 5.7with 1M KOH) 25 mg/l kanamycin was added if antibiotic selection oftransgenic seedlings was necessary.

These procedures were performed in a laminar flow hood.

Alternatively, for extended timelapse imaging of roots, sterile seedswere sown in coverslip based vessels (Nunc) which comprised 4 wells,each containing about 400 μl of low gelling temperature agarose with GMmedium. The roots of these plants grow down though the media and thenalong the surface of the coverslip. The roots are then ideallypositioned for high resolution microscopic imaging through the base ofthe vessel.

(4) Sealed plates or vessels were incubated for 1-3 days in the dark at4° C., and then transferred to an artificially lit growth room at 23° C.for germination.

(5) Arabidopsis seedlings germinate after 3 days, and can be used formicroscopy for several weeks. Root and shoot tissues can be directlyscored for GFP expression using an inverted fluorescence microscope(Leitz DM-IL) fitted with filter sets suitable for UV (Leitz-D;excitation filter 355-425 nm, dichroic mirror 455 nm, longpass emissionfilter 460 nm) and blue (Leitz-13; excitation filter 450-490 nm,dichroic mirror 510 nm, longpass emission filter 520 nm) lightexcitation of GFP. Roots, which grow along the base of the petri dishcan be observed directly by epifluorescence microscopy through the clearplastic base. Shoot tissues were directly observed in inverted dishes byusing one or two 7 mm threaded extension tubes with a 4× objective (EF4/0.12), that gave greater working distances. Epifluorescence imageswere captured in Adobe Photoshop using a Sony DXC-930P 3-chip CCD videocamera and F100-MPU integrating frame store, connected to a Nu Vista+video digitiser in an Apple Macintosh computer.

GFP-expressing Arabidopsis seedlings were removed from agar media, andsimply mounted in water under glass coverslips for microscopy. Growingroots could also be directly viewed through coverslip based vessels.Specimens were examined using a BioRad MRC-600 laser-scanning confocalmicroscope equipped with a 25 m W krypton-argon or argon ion laser andfilter sets suitable for the detection of fluorescein and texas red dyes(BioRad filter blocks K1/K2 with krypton-argon ion laser, and A1/A2 withargon ion laser). We routinely use a Nikon 60x PlanApo N. A. 1.2 waterimmersion objective to minimise loss of signal through sphericalaberration at long working distances. For the collection of timelapseimages, the laser light source was attenuated by 99% using a neutraldensity filter, the confocal aperture was stopped down and single scanswere collected at two second intervals. The large data files weretransferred to an Apple Macintosh computer, and the programs PicMergeand 4DTurnaround were used with Adobe Photoshop and Premiere to produceQuickTime movies for display and analysis.

GFP fluorescence can be seen from 4 days after Agrobacteriuminoculation, depending on the expression pattern. The plantletsexhibiting fluorescence can be used to construct a library oftransformed plants.

Example 7 Transactivation

mHAP1-VP16 expression within these lines can be used to direct theexpression of a chosen gene at a precise time and place within theorganism. The inventors have produced transgenic plants which maintainregulatory proteins or toxins, silent behind a HAP1-responsive promoter.These genes can now be activated in specific cells by crossing to achosen mHAP1-VP16 expressing line.

A stable transformed line HJR1 of Arabidopsis thaliana, which forms partof the library described above, expresses modified GFP (under theinfluence of the mHAP1-VP16 activator) in the cells of the extreme roottip. Similar lines have also been produced which carry a localised cyanfluorescent protein, driven by the mHAP1-VP16 gene.

Using standard techniques, the line is crossed with another Arabidopsisline which comprises a silently maintained GUS reporter gene underoperable control of a HAP1-responsive UAS. The plantlets obtained fromthe cross express GUS under the influence of the HAP1-VP16transcriptional activator. The pattern of expression is the same as thatfor the GFP reporter gene in the parent cell line (i.e. at the extremeroot tip). Thus the modified HAP1 DNA binding domain sequence is enablesthe expression of chosen genes of interest (e.g. GUS) in a predictablepattern and enables simultaneous expression of a plurality of genes ofinterest (e.g. GFP and GUS).

1. An isolated nucleic acid molecule comprising a modified HAP1DNA-binding domain nucleotide sequence encoding at least an effectiveportion of a HAP1 DNA-binding domain, characterised in that saidmodified nucleotide sequence has an A/T base content substantiallyreduced compared to the wild-type sequence such as to be expressible ina plant cell.
 2. A nucleic acid molecule as claimed in claim 1 whereinthe effective portion comprises amino acid residues 1 to 94 of the yeastHAP1 HAP1 polypeptide.
 3. A nucleic acid molecule as claimed in claim 1wherein the % A/T base content of the modified nucleotide sequence isless than 45%.
 4. A nucleic acid molecule as claimed in claim 1 whereinthe effective portion of a HAP1 DNA-binding domain has the amino acidsequence of FIG. 2 b (bottom line).
 5. A nucleic acid molecule asclaimed in claim 4 wherein the modified nucleotide sequence has thesequence of FIG. 2 b (middle line)
 6. A nucleic acid molecule as claimedin claim 1 wherein the modified nucleotide sequence is fused to a secondnucleotide sequence which encodes a transcriptional activator domainsuch as to encode a HAP1 transcriptional activator.
 7. A nucleic acidmolecule as claimed in claim 6 wherein the transcriptional activatordomain is selected from the activation domain of the HAP1 protein orherpes simplex virus (HSV) VP-16.
 8. A nucleic acid molecule as claimedin claim 7 wherein the modified nucleotide sequence and the secondnucleotide sequence encode the amino acid sequence of the mHAP1-VPI6chimera shown in FIG. 2 c (bottom line).
 9. A nucleic acid molecule asclaimed in claim 8 wherein the modified nucleotide sequence and secondnucleotide sequence consist of the sequence shown in FIG. 2 c (topline).
 10. A recombinant vector which comprises the nucleic acid ofclaim
 1. 11. A vector as claimed in claim 10 which is a plant vectorcomprising right and left Ti-DNA, to enable stable insertion into thegenome of a plant host cell.
 12. A vector as claimed in claim 10 whereinthe nucleic acid is operably linked to a promoter for transcription in ahost cell, wherein the promoter is optionally an inducible promoter. 13.A vector as claimed in claim 12 wherein the promoter is an enhancerdependent promoter such that if the promoter is inserted into a planthost cell genome in functional relationship with an enhancer sequenceand required transcription factors, the promoter will direct expressionin a tissue specific manner.
 14. A vector as claimed in claim 10 furthercomprising a reporter nucleotide sequence consisting of a reporter geneoperably linked to a HAP1 upstream activation sequence.
 15. A vector asclaimed in claim 14 wherein the reporter gene encodes a reporterpolypeptide capable of generating a visually detectable signal.
 16. Avector as claimed in claim 15 wherein the visually detectable signal canbe monitored by multispectral dynamic imaging.
 17. A vector as claimedin claim 15 wherein the reporter polypeptide is a wild-type of modifiedGFP.
 18. A vector as claimed in claim 17 wherein the modified GFP isencoded by mgfp5-ER.
 19. A vector as claimed in claim 17 wherein themodified GFP is a GFP extensin reporter gene fusion.
 20. A vector asclaimed in claim 19 wherein the GFP extensin reporter gene fusion is asclaimed in any one of claims 39 to
 42. 21. A composition of mattercomprising a pair vectors, which vectors are: (i) a vector as claimed inany one of claims 10 to 13, (ii) a vector comprising a nucleic acidwhich includes a reporter nucleotide sequence consisting of a reportergene operably linked to a HAP1 upstream activation sequence, whichreporter nucleotide sequence is as described in any one of claims 14 to20.
 22. A method which comprises the step of introducing the vector orpair of vectors of claim 10 into a host plant cell, and optionallycausing or allowing recombination between the vector or vectors and theplant cell genome such as to transform the host cell.
 23. A plant cellcontaining or transformed with the vector or pair of vectors of claim10.
 24. A method for producing a transgenic plant, which methodcomprises the steps of: (a) performing a method as claimed in claim 22,(b) regenerating a plant from the transformed plant cell.
 25. Atransgenic plant which is obtainable by the method of claim 24, or whichis a clone, or selfed or hybrid progeny or other descendant of saidtransgenic plant, which in each case includes a nucleic acid of any oneof claims 1 to
 9. 26. A plant as claimed in claim 25 which hasintegrated therein a reporter nucleotide sequence consisting of areporter gene operably linked to a HAP1 upstream activation sequencewhich reporter nucleotide sequence is as described in any one of claims14 to
 20. 27. A population of plants as claimed in claim 26 wherein theintegrated reporter gene differs between said plants, plantlets or partsthereof.
 28. A method comprising introducing a gene of interest into theplant or plants of claim 26 which gene of interest is operable linked toa HAP1 upstream activation sequence, such that the pattern of expressionof the gene of interest is the same as that of the reporter gene.
 29. Amethod as claimed in claim 28 wherein the gene of interest is introducedand thereby trans-activated by crossing.
 30. A method as claimed inclaim 28 wherein the gene of interest is introduced by means of a plantvector.
 31. A method of identifying a plant enhancer nucleic acidsequence, comprising the steps of: (i) transforming a plant cell hostwith a vector as claimed in claim 13, (ii) observing said pantexpression of said HAP1 binding domain, and (iii) optionally,characterising the position and\or nucleic acid sequence of the enhancersequence.
 32. A method of controlling expression of a gene of interestin a plant, the method comprising the steps of: (i) providing a plantcomprising a nucleic acid encoding a HAP1 transcriptional activator asdefined in claim 6 under the control of a naïve promoter such thatexpression of the HAP1 transcriptional activator is limited to thosecell types in which a naïve promoter sequence is in functionalrelationship with a host cell enhancer sequence and requiredtranscription factors, (ii) introducing the gene of interest into saidplant or part thereof, said gene of interest having an HAP1 responsiveupstream activation sequence, wherein binding of said HAP1transcriptional activator to said upstream activator sequence causestranscriptional activation of the gene of interest.
 33. A method asclaimed in claim 32 wherein the gene of interest encodes a toxin.
 34. Amethod as claimed in claim 32 wherein the gene of interest is of unknownfunction.
 35. A method of determining the function of a gene of interestcomprising the steps of: (i) performing a method as claimed in claim 34,(ii) comparing the phenotype of said plant or part thereof in which saidgene of interest is expressed with a second plant or part thereof inwhich said gene of interest is not expressed.
 36. A method ofindependently controlling expression of a first and a second gene ofinterest in a plant comprising the steps of: (i) providing a plantcomprising a nucleic acid encoding a HAP1 transcriptional activator asdefined in claim 6, and a second nucleic acid sequence, which encodes aGAL4 transcriptional activator; (ii) introducing the first gene ofinterest into a plant or part thereof, said first gene of interesthaving an HAP1 responsive upstream activation sequence; introducing thesecond gene of interest into a plant or part thereof, said second geneof interest having a GAL4 responsive upstream activation sequence;wherein binding of said HAP1 transcriptional activator to said upstreamactivator sequence causes transcriptional activation of the first geneof interest and binding of said GAL4 transcriptional activator to saidupstream activator sequence causes transcriptional activation of thesecond gene of interest.
 37. A method of coordinating the expression ofa plurality of genes of interest in a plant or part thereof, comprisingthe steps of: (i) providing a plant comprising a nucleic acid encoding aHAP1 transcriptional activator as defined in claim 6, and a secondnucleic acid sequence, which encodes a GAL4 transcriptional activator;(ii) introducing the genes of interest into a plant or part thereof,said genes of interest each being under the control of an HAP1responsive upstream activation sequence, wherein binding of said HAP1transcriptional activator to said upstream activator sequence causestranscriptional activation of the genes of interest.
 38. A method asclaimed in claim 37 wherein the plurality of genes are associated with asingle upstream activator sequence.
 39. An isolated nucleic acidmolecule comprising a reporter gene nucleotide sequence, which reportergene encodes a green fluorescent protein linked to an effective portionof extensin.
 40. A nucleic acid as claimed in claim 39 wherein theeffective portion comprises at least 60% of the full-length sequence ofa carrot extensin polypeptide.
 41. A nucleic acid as claimed in claim 40wherein the reporter gene nucleotide sequence encodes the amino acidsequence of FIG. 3 b (bottom line).
 42. A nucleic acid as claimed inclaim 41 wherein the reporter gene nucleotide sequence has the sequenceof FIG. 3 a (top line).
 43. A recombinant vector which comprises thenucleic acid of claim
 39. 44. A vector as claimed in claim 43 which is aplant vector comprising right and left Ti-DNA to enable stable insertioninto the genome of a plant host cell.
 45. A vector as claimed in claim43 wherein the nucleic acid is operably linked to a promoter fortranscription in a host cell, wherein the promoter is optionally aninducible promoter.
 46. A vector as claimed in claim 43 wherein thereporter gene is operably linked to a HAP1 upstream activation sequenceto form a reporter nucleotide sequence consisting.
 47. A method whichcomprises the step of introducing the vector of claim 43 into a hostplant cell, and optionally causing or allowing recombination between thevector and the plant cell genome such as to transform the host cell. 48.A plant cell containing or transformed with a vector of claim
 43. 49. Amethod for producing a transgenic plant, which method comprises thesteps of: (a) performing a method as claimed in claim 48, (b)regenerating a plant from the transformed plant cell.
 50. A transgenicplant which is obtainable by the method of claim 49, or which is aclone, or selfed or hybrid progeny or other descendant of saidtransgenic plant, which in each case includes a nucleic acid of any oneof claims 39 to
 42. 51. A method of cell sorting including the step ofscreening plants, plantlets, parts or cells thereof for expression of anucleic acid of claim 39, and selecting those plants, plantlets, partsor cells thereof which express said nucleic acid.
 52. An isolatedpolypeptide encoded by the nucleic acid of claim
 39. 53. An isolatedpolypeptide encoded by the nucleic acid of claim 1.